PROJECT SUMMARY BIOPHYSICAL PRINCIPLES OF MICROTUBULE DYNAMICS Microtubules are cytoskeletal polymers essential for cell division, cell motility and intracellular transport, and are implicated in many cancers and neurological disorders. Remodeling of the microtubule network architecture in space and time relies on the ability of individual microtubules to switch between periods of growth and shrinkage, behavior known as `microtubule dynamic instability'. Although discovered more than thirty years ago, the molecular mechanisms of microtubule dynamic instability and its regulation remain largely unknown. This problem is exacerbated by a complex and highly interconnected network of microtubule- associated proteins, which collectively regulate microtubule dynamics inside of cells. The goal of this project is to provide a fundamental molecular understanding of microtubule behavior and its regulation. To accomplish this goal, we will use an interdisciplinary approach, combining biology and physics. Inspired by conceptual models developed using cell biological tools, we will employ biochemical in vitro reconstitution with purified protein components, single-molecule total-internal-fluorescence (TIRF) imaging, and microfluidics techniques to obtain a quantitative description of the microtubule system behavior. Microtubule length distributions are determined by the combination of microtubule growth rates, shrinkage rates and the rates of transitions from growth to shrinkage (catastrophe) and back (rescue). However, these four parameters are only a manifestation of the underlying molecular mechanisms that define the macroscopic parameter values and their mutual relationships. We will address the key question of the coupling between microtubule growth and catastrophe by probing previously unattainable experimental regimes, critical for rigorous testing of existing and the development of new theoretical models. We will perform detailed measurements of the microtubule minus end dynamics, which to date remains largely unstudied, although recently recognized to be actively regulated inside cells. We will determine the molecular mechanisms of microtubule rescue, least well understood parameter of dynamic instability, which plays an important role in interphase microtubule architecture. Our measurements will be performed with purified tubulin, as well as ensembles of microtubule-associated proteins (including end-binding EB proteins, TOG-domain XMAP215 and CLASP proteins, and kinesin motor proteins from Kinesin-13 and -14 families) in order to elucidate their collective effects on microtubule behavior. Based on this quantitative characterization, we will develop predictive mathematical models that will capture the fundamental principles of microtubule dynamics and its regulation. The predictions of our quantitative models will be tested inside cells. This combination of theory and experiment will provide fundamental insight into microtubule system regulation, ultimately laying the foundation for new advances in human health.